PROJECT SUMMARY Cancer immunotherapy ? where the immune system is unleashed to destroy cancer cells ? is a revolutionary new approach for combating cancer. However, even though immunotherapy has achieved unprecedented efficacies in the clinic, many cancers are not yet amenable to immune therapy, and even for those that are, a majority of patients fail to respond. New agents with improved or complementary mechanisms of action are therefore needed. One intriguing, yet poorly understood potential cancer immunotherapy agent is the small molecule Val-boroPro. Val-boroPro induces immune-mediated tumor regressions in multiple mouse models of cancer by inducing pyroptosis, a lytic form of programmed cell death, in monocytes and macrophages. Notably, Val-boroPro was the first small molecule discovered that induces pyroptosis. It is now established that inhibition of the cytosolic serine peptidases DPP8 and DPP9 (DPP8/9) by VbP activates the pro-protein form of caspase-1 to mediate cell death, but how DPP8/9 inhibition leads to the activation of caspase-1 remains entirely unknown. The long-term goal of this project is to successfully harness this pathway for cancer immunotherapy. The objective of this proposal is to determine how the inhibition of DPP8/9 induces pyroptosis in monocytes and macrophages. The central hypothesis is that DPP8/9 cleave and inactivate one or more peptides, which, if not cleaved, activates the innate immune sensor protein Nlrp1. Activated Nlrp1 then activates pro-caspase-1, triggering pyroptosis. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory and described in the application. This hypothesis will be tested by pursuing three specific aims: 1) Determine strain, species, and cell type sensitivity to DPP8/9 inhibitors, as Nlrp1 is highly polymorphic and varies considerably across strains and species; 2) determine the molecular mechanism of Nlrp1 activation by DPP8/9 inhibitors; and 3) determine the function of a specific co- chaperone required for DPP8/9 inhibitor-induced pyroptosis, which we hypothesize is responsible for the stability, folding, or trafficking of a key pyroptosis factor (e.g., Nlrp1 or the DPP8/9 substrate). Successful completion of this proposal will uncover new principles and mechanisms regulating the innate immune system, providing insights into pathogen recognition, autoimmune disorders, and inflammation. Moreover, this work has extraordinary potential to identify new targets and strategies that could form the bases of novel cancer immunotherapies.